A weak African swine fever strain with large gene fragment deletion in multiple gene families and its construction method and application

Abstract

The present invention provides a weak African swine fever strain with a large gene fragment deletion in a multi-gene family, and a construction method and application thereof. The provided natural weak strain, compared with the strong African swine fever strain, has a nucleic acid fragment between MGF110-9L and MGF360-14L in the left variable region of the genome deletion. The present invention also provides a method for constructing a weak African swine fever virus strain, which is to construct a weak strain after removing the nucleic acid fragment between MGF110-9L and MGF360-14L in the left variable region of the strong African swine fever virus strain. The natural deletion strain and the artificially constructed gene deletion strain provided by the present invention both have large-scale deletions in the core region of the multi-gene family, and the strain is genetically stable, without recombination and mutation, so the strain has a natural attenuating property, which is of great significance for basic research and vaccine development.

Description

Technical Field

[0001] The present invention belongs to the technical field of constructing attenuated African swine fever virus strains, and specifically relates to an attenuated African swine fever strain with a large gene fragment deletion in a multi-gene family, and a construction method and application thereof. Background Art

[0002] African swine fever (ASF), caused by the African swine fever virus (ASFV), is a highly contagious, lethal infectious disease of domestic and wild pigs. Symptoms can manifest in four forms: acute, acute, subacute, and chronic. Pigs infected with ASFV develop fever, hyperviremia, and hemorrhagic lesions. Some strains can cause high morbidity and mortality. The clinical symptoms and pathological changes of the disease are very similar to those of swine fever.

[0003] The ASFV genome is a linear, double-stranded DNA construct, approximately 170 to 190 kb in length. It consists of a left variable region (LVR) of 38 to 48 kb, a central conserved region (C region) of approximately 125 kb, and a right variable region (RVR) of 13 to 22 kb. Different strains may differ in key locations, such as the multigene family (MGF) within the LVR, the central variable region (CVR) within the C region, and the EP402R gene (which expresses the CD2v protein).

[0004] Since the disease was first reported in 1921, it has attracted attention due to its highly infectious characteristics. However, due to objective reasons such as the large viral genome, multiple genotypes, complex infection mechanism and the fact that some regions are very easy to mutate, no effective vaccine has been available so far.

[0005] It has been confirmed that the MGF360 gene and MGF505 gene located in the variable region at the left end of the ASFV genome are related to viral replication, viral virulence and the host range of the virus. Typical natural attenuated strains such as NH / P68 (isolated from chronically infected pigs in Portugal in 1968) and OURT 88 / 3 (isolated from Ornithodoros ticks wandering in pig farms in Portugal in 1988) both experienced gene loss in the MGF360-10L to MGF505-2R region (the deletion was approximately 10Kbp in length); cell-passaged attenuated strains such as BA71V (the BA71 strain isolated in Spain in 1971 was passaged on bone marrow cells for 36 generations and then adapted to Vero cells for 100 generations) and MS16 (the E70 strain isolated in Spain in 1971 was passaged on leukocytes for 6 generations and then adapted to MS monkey kidney cells for 16 generations) experienced gene loss in the MGF360-10L (A356L) to MGF505-4R (A505R) region (the deletion was approximately 8Kbp in length). Artificially constructed knockout strains have also confirmed that deletions of multiple gene families, including the MGF360 / 505 genes (approximately 8 kbp in length), MGF360-18R, MGF110-9L, and MGF505-7R genes, can also attenuate viral virulence. However, concerns about residual virulence or reversion to virulence in attenuated live vaccines have led to particularly cautious and demanding evaluations for ASF vaccine development. Therefore, continued exploration of the functions of ASF multigene families and the impact of deletions of larger segments of these families are crucial and a practical need for the development of new ASF vaccines. Summary of the Invention

[0006] The purpose of the present invention is to provide an ASF attenuated strain with a large gene fragment deletion in a multi-gene family, and its construction method and application; the prepared deletion strain can be used to prepare a vaccine to prevent diseases caused by ASFV.

[0007] The present invention first provides an ASF natural attenuated strain (abbreviated as CN2018-LVR strain in the specification) with a large gene fragment deletion in a multi-gene family. Compared with the ASF virulent strain, the genome of the natural attenuated strain lacks the gene fragments from MGF110-9L to MGF360-14L in the left variable region.

[0008] The present invention also provides a method for constructing an ASFV attenuated strain, which comprises removing the gene fragment between MGF110-9L and MGF360-14L in the left variable region of the ASFV virulent strain and then constructing the attenuated strain;

[0009] More specifically, the construction method is to obtain an artificial weak strain by knocking out the nucleic acid fragment between 12287bp and 32270bp of the genome of the ASFV strong strain;

[0010] The virulent strain, as a specific record in the embodiment, is ASFV China / LN / 2018 / 1 (GenBank accession number OP856591, abbreviated as CN2018);

[0011] In the method, the gene fragment is removed by homologous recombination.

[0012] The present invention also provides a recombinant plasmid for preparing an artificial attenuated strain, which is prepared by using 600 bp sequences on both sides of the deletion position corresponding to the large-scale natural deletion strain (CN2018-LVR strain) as upstream and downstream homology arms, and sequentially adding a P72 promoter, an mCherry sequence, and a NOS terminator sequence to the upstream homology arm sequence. The obtained sequence is synthesized and cloned into a homologous recombination vector for preparation;

[0013] In another aspect, the present invention further provides an artificial attenuated strain of ASFV, which is constructed using the above method;

[0014] The present invention also provides the use of the attenuated strain in preparing a vaccine;

[0015] The vaccine can be a live vaccine or an inactivated vaccine.

[0016] The present invention also provides a method for detecting attenuated ASF strains, wherein the method is to detect whether the above-mentioned deletion of the nucleic acid fragment of 12287 bp to 32270 bp exists in ASFV;

[0017] The detection method, wherein the primer sequences used are as follows:

[0018] CN2018-LVR PCR-F: 5′-GCACCAGAACTTGAGAT-3′,

[0019] CN2018-LVR PCR-R: 5′-CCGTGAAGAGCGATA-3;

[0020] The present invention further provides a qPCR method for detecting the above-mentioned attenuated strains, wherein the primer / probe sequences used are as follows:

[0021] CN2018-LVR qPCR-F: 5′-CATGATGTGAAAGCGGCGTA-3′,

[0022] CN2018-LVR qPCR-R: 5′-TCTATTGGCGGGTAGCTTGT-3′,

[0023] CN2018-LVR qPCR-P: 5′-FAM-TAGCGAGAAACCCTACATA

[0024] T-MGB-3′; or

[0025] MGF 360-13L qPCR-F: 5′-TCTTTGGCGCCTAGCTGTCT-3′,

[0026] MGF 360-13L qPCR-R: 5′-GGCGCAAATATCAACTATGGTTT-3′,

[0027] MGF 360-13L qPCR-P: 5′-FAM-TCTCGGATGTGCTTCGT-BHQ-3′; or

[0028] MGF 110-11L qPCR-F: 5′-TCCCAGCAGAAATCACAGTGTT-3′,

[0029] MGF 110-11L qPCR-R: 5′-TTACCAAGGACTTATACGCCTCCTA-3′, MGF 110-11L qPCR-P: 5′-FAM-CCATACGTGCACCAGTAT-BHQ-3′.

[0030] The natural deletion strain and artificially constructed gene deletion strain provided by the present invention both have large-scale deletions in the core region of the multi-gene family, and the strains are genetically stable without recombination and mutation. Therefore, the strain has natural attenuating properties, which is of great significance for basic research and vaccine development. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 : CN2018-LVR strain-specific qPCR identification graph, where the positive curve represents the CN2018-LVR strain and the negative curve represents the CN2018 strain;

[0032] Figure 2 : qPCR identification of the genes on both ends of the deletion site of the CN2018-LVR strain, where the positive curve is the CN2018 strain and the negative curve is the CN2018-LVR strain;

[0033] Figure 3 : Electrophoresis of PCR amplification of the deletion position of the CN2018-LVR strain, where M: DNA Marker DL2 000; 1: CN2018-LVR strain; 2: CN2018 strain; 3: negative control;

[0034] Figure 4: Electrophoresis of PCR amplification of the virus deletion positions of the five generations of CN2018-LVR strain, where M: DNA Marker DL2 000; 1: F1 generation; 2: F2 generation; 3: F3 generation; 4: F4 generation; 5: F5 generation; 6: maternal strain control CN2018 strain;

[0035] Figure 5 : Red fluorescence photos observed under a fluorescence microscope, where the left image is a red fluorescence observation image (100 times), and the right image is a white light observation image (100 times);

[0036] Figure 6 : qPCR identification of the genes on both ends of the deletion site of the CN2018-ΔLVR strain, where the positive curve is the CN2018 strain; the negative curve is the CN2018-ΔLVR strain;

[0037] Figure 7 : PCR amplification electrophoresis of the deletion position of the CN2018-ΔLVR strain, where M1: DNA Marker DL10 000; M2: DNA Marker DL2 000; 1: CN2018 strain; 2: CN2018-ΔLVR strain;

[0038] Figure 8 : Electrophoresis diagram of PCR amplification of the virus deletion positions of five generations of CN2018-ΔLVR strain, where M: DNA Marker DL2 000; 1: F1 generation; 2: F2 generation; 3: F3 generation; 4: F4 generation; 5: F5 generation; 6: maternal strain control CN2018 strain. DETAILED DESCRIPTION

[0039] The present invention screened and obtained a naturally attenuated ASF strain ASFV China / LN / 2018 / 1-LVR (abbreviated as CN2018-LVR strain), and compared the strain sequence with the whole genome sequence of the ASF virulent strain. It was found that the CN2018-LVR strain did not contain the sequence between 12289bp (located in the MGF110-9L gene) and 32273bp (located in the MGF360-14L gene) of the virulent strain ASFV China / LN / 2018 / 1 (GenBank accession number OP856591), with a total deletion of 19985bp. That is, the naturally attenuated strain lacks a nucleic acid fragment totaling 19985bp from MGF110-9L (starting at 12289bp) to MGF360-14L (ending at 32273bp) in the left variable region.

[0040] Specifically, the naturally attenuated strain is an ASFV lacking MGF110-9L, MGF110-11L, MGF 110-14L, MGF 110-12L, MGF 110-13L, MGF 360-4L, MGF 360-6L, X69R, MGF 300-1L, MGF 300-4L, MGF 360-8L, MGF 360-9L, MGF 360-10L, MGF 360-11L, MGF 505-1R, MGF 360-12L, MGF 360-13L and MGF360-14L genes. Wherein MGF110-9L and MGF360-14L represent two genes of multigene family 110 and multigene family 360.

[0041] Among them, the multigene family (MGF) is a large number in the ASFV genome (accounting for 30% of the entire genome), mainly including MGF 100, MGF 110, MGF 300, MGF 360 and MGF 505. Each family is named according to the number of amino acids edited by the open reading frame. For example, MGF110-9L means that this gene encodes approximately 110 amino acids and is approximately the 9th gene of this type starting from the left side of the ASF genome. The coding region is translated from the 3' end to the 5' end (L). MGF360-14L means that this gene encodes approximately 360 amino acids and is approximately the 14th gene of this type starting from the left side of the ASF genome. The coding region is translated from the 3' end to the 5' end (L).

[0042] The ASF virulent strain (ASFV China / LN / 2018 / 1, abbreviated as CN2018) was isolated from the first ASF outbreak in Shenyang, Liaoning Province in 2018. 50 Inoculation of healthy domestic pigs resulted in the death of all vaccinated pigs within 2 weeks.

[0043] Based on the above findings, the present invention also provides a method for preparing an artificially deleted ASFV strain, which is a weak strain constructed by removing the nucleic acid fragment (12289 bp to 32273 bp) between MGF110-9L and MGF360-14L in the left variable region of the ASF strong strain.

[0044] For the above-mentioned gene deletion, in a specific embodiment of the present invention, homologous recombination technology is used, with the pUC57 plasmid as the vector and the ASF virulent strain CN2018 as the parent strain, to construct a recombinant plasmid containing the left and right homologous arms of the CN2018-LVR deletion position and red fluorescent protein (mCherry).

[0045] The recombinant plasmid was transfected into porcine alveolar macrophages (PAM cells) inoculated with the CN2018 strain for homologous recombination. After multiple rounds of plaque purification screening, quantitative PCR (qPCR), and PCR detection, a recombinant attenuated virus with extensive gene deletions was purified and named ASFV China / LN / 2018 / 1-ΔLVR strain (abbreviated as CN2018-ΔLVR strain).

[0046] The present invention is described in detail below with reference to the embodiments and accompanying drawings.

[0047] Example 1: Discovery and purification of a strain with a large-scale natural deletion of a multigene family (CN2018-LVR strain)

[0048] During the construction and purification of the ASFV vaccine candidate, the inventors conducted qPCR testing of ASFV commonly deleted genes in cell culture and unexpectedly discovered that the ASFV in the cell culture had a large gene deletion. Subsequently, using the limiting dilution method, the deleted virus was gradually diluted and purified and whole-genome sequencing was performed. The results confirmed that the strain had a large-scale gene loss in the MGF 110-9L to MGF 360-14L region of the left variable region (the deletion length was approximately 20Kb), without artificially modified components or inserted genes. The specific deleted genes include MGF110-9L (partial deletion), MGF110-11L, MGF 110-14L, MGF 110-12L, MGF 110-13L, MGF 360-4L, MGF 360-6L, X69R, MGF 300-1L, MGF 300-4L, MGF360-8L, MGF 360-9L, MGF 360-10L, MGF 360-11L, MGF 505-1R, MGF 360-12L, MGF 360-13L and MGF 360-14L (partial deletion). After virus purification, PCR, qPCR and virus titration were performed, and the results showed that the virus titer was 7.0HAD 50 / ml, and no gene loss or insertion occurred in the middle of the deletion position. This strain was named CN2018-LVR and stored at -80℃ for future use.

[0049] 1. qPCR identification

[0050] qPCR was used to detect the sequences at both ends of the large-scale deletion of the multigene family in the CN2018-LVR strain (MGF 110-11L and MGF 360-13L gene locations) and the entire large-scale deletion region. DNA was extracted using a nucleic acid extraction kit (Tianlong Technology). A 20-μl qPCR system was used: 10 μl of 2×AceQ U+Probe Master Mix (Novozymes), 0.4 μl of upstream and downstream primers (both 10 μM, sequences shown in Table 1), 0.2 μl of probe (10 μM), 2 μl of DNA template, and sterile deionized water to 20 μl.

[0051] Table 1: CN2018-LVR strain-specific qPCR detection primers and probes

[0052]

[0053]

[0054] The reaction conditions were: 50°C for 2 minutes, 95°C for 5 minutes, 95°C for 15 seconds, 58°C for 1 minute, and 45 cycles of amplification. qPCR results showed that the virus had a specific amplification curve ( Figure 1 ) and no amplification curve was found in the qPCR detection of MGF 360-13L gene and MGF 110-11L gene ( Figure 2 ), proving that the multi-gene family of the virus has lost a large range of positional sequences correctly, without recombination or mutation.

[0055] 2. PCR identification

[0056] PCR was used to detect large-scale deletions in multiple gene families in the CN2018-LVR strain. DNA was extracted from the test sample using a nucleic acid extraction kit (Tianlong Technology). A 50-μl PCR system was used: 25 μl of 2×Gflex PCR Buffer (Takara Biotech), 2 μl of upstream and downstream primers (both 10 μM), 1 μl of TksGflex DNA Polymerase (1.25 U / μl), 2 μl of DNA template, and sterile purified water to 50 μl.

[0057] Table 2: Primer information for detecting large deletion sites in the CN2018-LVR strain

[0058]

[0059] The reaction conditions were: 94°C for 1 minute; 98°C for 10 seconds, 52°C for 15 seconds, and 68°C for 1 minute, for 35 cycles. After the PCR reaction, the product was electrophoresed on a 1.0% agarose gel to detect the amplification results and photographed. The electrophoresis results showed that the specific target fragment size was 846 bp, which was consistent with the expected fragment size, and there were no other non-specific bands ( Figure 3 ). Sequencing and comparison analysis showed that the sequences of the multi-gene family of the virus were correct in the large-scale loss positions, and no recombination or mutation occurred.

[0060] 3. Virus titration and identification

[0061] The virus was serially diluted 10-fold using DMEM cell maintenance medium containing 2% fetal bovine serum. -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 Five dilutions were inoculated into PAM cells (96-well cell culture plates) with more than 80% adherence. Each dilution was inoculated into 8 wells, with 0.1 ml per well. The cells were cultured at 37°C and 5% CO2. On the day of virus inoculation, 0.02 ml of fresh 1% porcine erythrocytes prepared in sterile PBS (0.01 mol / L, pH 7.2-7.4) was added to each well. The erythrocyte adsorption was observed microscopically daily for 5-7 consecutive days. Patients with erythrocyte adsorption were considered infected and the HAD was calculated. 50 The results showed that the virus content was 7.0HAD 50 / ml.

[0062] 4. Genetic stability identification

[0063] To evaluate the genetic stability of the CN2018-LVR strain in PAM cells, the CN2018-LVR strain was propagated in PAM cells for 5 generations. 50 , evaluate its reproduction stability; evaluate the stability of its gene deletion by PCR determination of the large-scale deletion position of each generation of virus. 50 The determination refers to 1.3, and the PCR determination refers to 1.2. The results show that the virus can be stably reproduced in each generation, and the virus titer is 10 6.7 ~10 7.0 TCID 50 / ml (Table 3); PCR amplification electrophoresis results showed that the specific target fragment size of each generation virus was 846 bp, consistent with the expected fragment size, and no other non-specific bands ( Figure 4 ), sequencing results showed that the sequences on both sides of the large-scale deleted genes in the CN2018-LVR strain were correct, and no gene loss or insertion occurred in the middle of the deleted sequence.

[0064] Table 3: Virus content determination results of 5 passages of CN2018-LVR strain

[0065]

[0066]

[0067] Example 2: Safety evaluation of a large-scale naturally deleted strain of a multigene family (CN2018-LVR)

[0068] To understand the safety of CN2018-LVR strain inoculation in healthy susceptible pigs. 6.0 HAD 50 Five healthy susceptible pigs were inoculated with 100 mg / ml of virus solution and observed for 21 days after inoculation. The safety of overdose inoculation of this strain was evaluated through clinical observation, body temperature monitoring, detoxification and other safety indicators.

[0069] 1. Clinical indicator observation results

[0070] The status of the vaccinated pigs was observed daily after vaccination. The results showed that all pigs were in good spirits, ate normally, had no respiratory abnormalities such as coughing, wheezing, and difficulty breathing, no digestive system abnormalities such as vomiting and diarrhea, and no adverse reactions in the joints, skin, conjunctiva, or injection site (Table 4).

[0071] Table 4: Clinical manifestation observation table

[0072]

[0073] 2. Body temperature monitoring results

[0074] Body temperatures were measured daily for 21 days after vaccination to verify safety. Results showed that body temperatures were normal over the 21 days, with no temperature exceeding 40.5°C. (See Table 5 for details.)

[0075]

[0076]

[0077] Table 5: Body temperature measurement record after CN2018-LVR vaccination (℃)

[0078]

[0079] 3. Oral, nasal and anal detoxification monitoring results

[0080] After vaccination, oral, nasal, and anal swabs were collected on days 1, 3, 5, 7, 10, 14, and 21, and tested by qPCR (Qingdao Lijian Biotechnology Co., Ltd., ASFV fluorescent PCR detection kit), and the results were all negative, indicating no oral, nasal, or anal excretion (Tables 6, 7, and 8).

[0081] Table 6: Oral detoxification status (qPCR method)

[0082]

[0083] Note: “-” indicates negative;

[0084] Table 7: Nasal detoxification status (qPCR method)

[0085]

[0086] Note: “-” indicates negative;

[0087] Table 8: Anal detoxification status (qPCR method)

[0088]

[0089] Note: “-” indicates negative;

[0090] These results demonstrate that high-dose inoculation of healthy, susceptible pigs with the CN2018-LVR strain resulted in good mental state, normal feeding, and no respiratory abnormalities such as coughing, wheezing, or dyspnea during the 21-day observation period. There were no digestive system abnormalities such as vomiting or diarrhea, and no adverse reactions to the joints, skin, conjunctiva, or injection site. Daily temperature measurements showed no significant temperature elevation. No evidence of toxin excretion was observed in oral, nasal, or anal samples collected 1, 3, 5, 7, 10, 14, and 21 days after inoculation. Therefore, high-dose inoculation of healthy, susceptible pigs with the CN2018-LVR strain is safe.

[0091] Example 3: Evaluation of the protective effect of the challenge with a large-scale naturally deleted strain of a multigene family (CN2018-LVR)

[0092] On the 21st day of the CN2018-LVR safety test, 5 immunized pigs and 3 blank control pigs were intramuscularly injected with 1 ml / head of ASF virulent CN2018 strain (100 HAD 50 After challenge, the mice were observed for 28 consecutive days, with daily observation of clinical symptoms, temperature measurement, and statistics of survival, morbidity, and mortality in each group.

[0093] 1. Clinical indicator observation results

[0094] Within 28 days after challenge, all three pigs in the blank control group died on day 8. The five pigs immunized with the CN2018-LVR strain maintained a stable mental state, ate normally, and showed no respiratory abnormalities such as coughing, wheezing, or dyspnea. There were no digestive system abnormalities such as vomiting or diarrhea, and no adverse reactions to the joints, skin, conjunctiva, or injection site.

[0095] 2. Body temperature monitoring results

[0096] Within 28 days after challenge, all three pigs in the blank control group developed fever on day 4 after challenge and died. The five pigs immunized with the CN2018-LVR strain did not experience a significant increase in body temperature throughout the entire process (Table 9).

[0097] Table 9: Temperature measurement record of CN2018-LVR vaccinated pigs after virus challenge (℃) Note: “ / ” indicates death

[0098]

[0099]

[0100]

[0101] Results showed that within 28 days after challenge, all five pigs in the control group died, while all five pigs immunized with the CN2018-LVR strain survived. During the 28-day observation period, the pigs remained in good spirits, ate normally, and showed no respiratory abnormalities such as coughing, wheezing, or dyspnea. There were no digestive system abnormalities such as vomiting or diarrhea. There were no adverse reactions to the joints, skin, conjunctiva, or injection site. Daily temperature measurements showed no significant temperature elevation. These findings suggest that pigs immunized with the CN2018-LVR strain provide adequate protection against challenge with the virulent ASF strain.

[0102] Example 4: Construction and purification of artificial deletion strain (CN2018-ΔLVR strain)

[0103] To further verify the stability of the deletion position of the large-scale natural attenuated strain obtained by the present invention, the inventors constructed a recombinant plasmid pUC-LVR-mCherry containing homologous arms on both sides of the deletion position of the large-scale natural attenuated strain (CN2018-LVR strain) and a red fluorescent protein. The pUC-LVR-mCherry recombinant plasmid was transfected into porcine alveolar macrophages (PAM cells) inoculated with the ASF virulent strain (CN2018) for homologous recombination. After multiple rounds of plaque purification and screening, the screened recombinant viruses were subjected to qPCR, PCR detection, and sequencing analysis, confirming that the obtained virus was a large-scale artificial deletion strain, named CN2018-ΔLVR strain.

[0104] The steps for constructing the CN2018-ΔLVR strain are described below.

[0105] 1. Construction of homologous recombination plasmid

[0106] A 600-bp sequence flanking the deletion site of a large-scale naturally attenuated strain (CN2018-LVR strain) was selected as the homology arms. The P72 promoter, mCherry sequence, and NOS terminator sequence were sequentially added to the homology arms. This sequence was synthesized and cloned into the pUC57 vector. The homologous recombinant plasmid was named pUC-LVR-mCherry.

[0107] 2. Construction, transfection, and purification of CN2018-ΔLVR strain

[0108] More than 80% of the PAM cells (6-well cell culture plates) that were attached were inoculated with the CN2018 strain at a dose of MOI=3 (multiplicity was added in sequence, respectively for the recombinant viruses to be screened), and then the homology arm recombinant plasmid pUC-LVR-mCherry was transfected using FuGENE HD TransfeCtionReagent. On the third day after transfection, the PAM cells that showed red fluorescence were collected, frozen and thawed once, and the supernatant was diluted in a certain proportion and inoculated with more than 90% of the PAM cells that were attached. After culturing for 3 days, the fluorescence was observed and screened according to the plaque purification method. After multiple rounds of purification, the number of red fluorescence cells observed under a fluorescence microscope was close to the entire observation field ( Figure 5 ).

[0109] 3. qPCR identification of CN2018-ΔLVR strain

[0110] Wells with high red fluorescence were selected, and qPCR was used to detect the two inner genes (MGF 110-11L and MGF 360-13L genes) of the CN2018-ΔLVR strain, which has a large-scale deletion of the multigene family. DNA of the test sample was extracted using a nucleic acid extraction kit (Tianlong Technology). A 20μl qPCR system was used: 10μl of 2×AceQ U+Probe Master Mix (Novozymes), 0.4μl of upstream and downstream primers (both 10μM), 0.2μl of probe (10μM), 2μl of DNA template, and sterile deionized water was used to make up to 20μl.

[0111] Table 10: CN2018-ΔLVR strain-specific qPCR detection primers and probes

[0112]

[0113] The reaction conditions were: 50°C for 2 minutes, 95°C for 5 minutes, pre-denaturation at 95°C for 15 seconds, 58°C for 1 minute, and 45 cycles of amplification. qPCR results showed no specific amplification curves for the MGF 360-13L and MGF 110-11L genes in the CN2018-ΔLVR strain, indicating that a large range of target genes had been deleted ( Figure 6 ).

[0114] 4. PCR identification of CN2018-ΔLVR strain

[0115] The PCR method was used to detect the gene deletion position of the CN2018-ΔLVR strain. The DNA of the sample to be tested was extracted using a nucleic acid extraction kit (Tianlong Technology), and a 50μl PCR system was used: 2×Gflex PCR Buffer (Bao Biotechnology) 25μl, 2 μl of upstream primers and downstream primers (both 10μM), 1μl of TksGflex DNA Polymerase (1.25U / μl), 2μl of DNA template, and sterile purified water to 50μl. The reaction conditions were: 94°C for 1 minute; 98°C for 10 seconds, 52°C for 15 seconds, 68°C for 2 minutes, and 35 cycles of amplification. After the PCR reaction was completed, the product was subjected to 1.0% agarose gel electrophoresis to detect the amplification results and photographed. The electrophoresis results showed that the specific target fragment size was 2136bp, which was consistent with the expected fragment size, and there were no other non-specific bands ( Figure 7 ). Sequencing and comparison analysis showed that the sequences of the viral gene knockout and insertion were correct.

[0116] 5. Genetic stability identification

[0117] To evaluate the genetic stability of CN2018-ΔLVR strain in PAM cells, the CN2018-ΔLVR strain was propagated in PAM cells for 5 generations, and the HAD of the virus was measured at each generation. 50 , evaluate its reproduction stability; evaluate the stability of its gene deletion by PCR determination of the virus deletion position in each generation. 50 The determination refers to 1.3, and the PCR determination refers to 1.2. The results show that the virus can be stably reproduced in each generation, and the virus titer is 10 6.5 ~10 7.0 TCID 50 / ml (Table 11);

[0118] Table 11: Virus content determination results of 5 passages of CN2018-ΔLVR strain

[0119]

[0120] The results of PCR amplification and electrophoresis showed that the specific target fragment size of each generation of virus was 2136 bp, which was consistent with the expected fragment size, and there were no other non-specific bands ( Figure 8), sequencing results showed that the sequences on both sides of the deleted gene of the CN2018-ΔLVR strain were correct, and no gene insertion occurred in the middle of the deleted sequence. The artificially constructed CN2018-ΔLVR strain has a similar deleted sequence to the naturally attenuated strain CN2018-LVR strain, and the deleted sequence has been shown to contain important virulence genes of ASFV (such as MGF110-9L, MGF 505-1R, MGF 360-12L, MGF 360-13L and MGF 360-14L, etc.), which shows that the CN2018-ΔLVR strain has the characteristics of a attenuated strain.

[0121] 6. Safety and effectiveness verification

[0122] To determine the safety and efficacy of CN2018-ΔLVR inoculation of healthy susceptible pigs, 1.0 ml of 10 6.0 HAD 50 One healthy susceptible pig was inoculated with 100 mg / ml of virus solution and observed for 21 days after inoculation. The safety of overdose inoculation of this strain was evaluated through clinical observation, body temperature monitoring, and detoxification. Results: After inoculation, the pigs were in good spirits, had normal feeding habits, showed no respiratory abnormalities such as coughing, wheezing, or dyspnea, no digestive system abnormalities such as vomiting or diarrhea, and no adverse reactions to the joints, skin, conjunctiva, or injection site. Their body temperatures remained normal for 21 days, with none exceeding 40.5°C. After inoculation, oral, nasal, and anal swabs were collected on days 1, 3, 5, 7, 10, 14, and 21. qPCR testing (Qingdao Lijian Biotechnology Co., Ltd., ASFV Fluorescent PCR Detection Kit) revealed negative results, indicating no oral, nasal, or anal detoxification. In summary, it was determined that high-dose inoculation of healthy susceptible pigs with the CN2018-ΔLVR strain is safe.

[0123] On the 21st day of the CN2018-ΔLVR safety test, 1 ml / head (100 HAD) of ASF virulent CN2018 strain was injected intramuscularly. 50 After challenge, the pigs were observed for 28 consecutive days, with daily observations for clinical symptoms and temperature measurements. Survival, morbidity, and mortality were recorded for each group. Results: The vaccinated pigs remained mentally stable, had normal feeding habits, showed no respiratory abnormalities such as coughing, wheezing, or dyspnea, nor digestive system abnormalities such as vomiting or diarrhea. There were no adverse reactions to the joints, skin, conjunctiva, or injection site, and the vaccinated pigs showed no significant temperature increases. These findings indicate that the CN2018-ΔLVR strain protects pigs against challenge with virulent ASF strains.

Claims

1. A method for constructing a weak strain of African swine fever virus, characterized in that: The method is to remove the nucleic acid fragment between MGF110-9L and MGF360-14L in the left variable region of the virulent African swine fever virus to construct a weak strain; that is, the genome is missing a nucleic acid fragment of 12287 bp to 32270 bp; The virulent strain of African swine fever virus is ASFV China / LN / 2018 / 1.

2. The method according to claim 1, wherein The method is to remove nucleic acid fragments by homologous recombination.

3. An artificially attenuated strain of African swine fever virus, characterized in that: The artificial attenuated strain is constructed by the method described in claim 1.

4. Use of the artificially attenuated strain of African swine fever virus according to claim 3 in the preparation of vaccines.

5. An African swine fever virus vaccine, characterized in that: The antigen in the vaccine is the artificial attenuated strain of African swine fever virus as described in claim 3.

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